Project Summary Vertebrate vision and olfaction depend on cyclic nucleotide-gated (CNG) channels. These channels transduce chemical signals to electrical signals in photoreceptors and olfactory receptor cells. Mutations in CNG channel genes are associated with visual disorders such as retinitis pigmentosa and achromatopsia. CNG channels exhibit interesting and physiologically important properties; for example, their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage, they conduct Na+ and Ca2+ but are also blocked by external Ca2+, their activation gate is located in the ion selectivity filter rather than the cytoplasmic end of the ion conduction pathway. Despite extensive functional studies, to date, there is no high-resolution structure of a full-length CNG channel, limiting our understanding of CNG channel mechanisms and functions. In preliminary studies, we have solved a 3.5 -resolution single-particle cryo-electron microscopy (cryo-EM) structure of a full-length CNG channel from C. elegans, named TAX-4, in the cGMP-bound open state. This structure provides significant insights into CNG channel gating, ion permeation and channelopathy. We will build on this breakthrough and pursue the following studies: (1) We will obtain an unliganded, closed state TAX-4 structure. (2) Our new structure reveals an interface between a cGMP-controlled cytoplasmic gating ring and the transmembrane domain of the channel. We will study the importance and mechanistic contribution of this interface to TAX-4 gating by cGMP. We will also generate a mutant channel that can still bind cGMP but is fully closed, and solve the structure of this cGMP-bound closed channel, which will shed significant light on the mechanisms and structural changes involved in ligand gating. (3) Our newly obtained structure allows us to map the 3-D locations of many single amino acid missense mutations that cause retinitis pigmentosa and achromatopsia. We will select a set of mutations that we suspect may affect cGMP gating but not cGMP binding and examine the functional properties of the corresponding mutant channels, including membrane trafficking and gating by cGMP. As in aim 2, we will also select a mutant channel that can still bind cGMP but is fully closed, and solve the structure of this cGMP-bound closed channel, illustrating how a disease-causing mutation changes channel structure. (4) Our structure reveals that the ion selectivity filter is lined by the carboxylate side chains of a glutamate residue and three rings of backbone carbonyls. We will study the functional and structural impact of mutating this glutamate. We will also solve the structure of WT and mutant TAX-4 in Ca2+-containing solutions, providing insights to where and how Ca2+ binds in the selectivity filter. These studies will greatly enhance our knowledge on the mechanisms of CNG channel ion permeation, gating and channelopathy, and provide a framework for understanding cyclic nucleotide modulation of related channels, such as hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels.